1. Field of the Invention
The present invention relates to human neutral sphingomyelinases (N-SMases), including recombinant N-SMases and fragments and derivatives thereof and isolated nucleic acids encoding N-SMases and fragments and derivatives. In preferred aspects, assays for identifying compounds that can modulate N-SMase related activity are provided, particularly assays to identify a pharmacological agent useful in the diagnosis or treatment of disorders associated with human neutral sphingomyelinases.
2. Background
Sphingomyelinases type-C (E.C. 3.1.4.12) are a group of phospholipases that catalyze the hydrolytic cleavage of sphingomyelin via the following reaction (1). EQU Sphingomyelin.fwdarw.Ceramide+Phosphocholine (1)
Native N-SMase purified from human urine and cultured human kidney proximal tubular cell membranes has an apparent molecular weight of 92 kDa, neutral pH optima, is heat unstable and is localized on the surface of various cells. S. Chatteijee, Adv. Lipid Res., 26:25-48 (1993); S. Chatterjee et al., J. Biol. Chem., 264:12,534-12,561 (1989); and S. Chatterjee et al., Methods in Enzymology, Phospholipase, 197:540-547 (1991). N-SMase action has been shown to mediate signal transduction of vitamin D.sub.3, tumor necrosis factor-.alpha. (TNF-.alpha.), interferon-gamma and nerve growth factor (Y. Hannun, J. Biol. Chem., 269:3,125-3,128 (1994); S. Chatterjee, J. Biol. Chem., 268:3,401-3,406 (1993); and S. Chatterjee, J. Biol. Chem., 269:879-882 (1994)) leading to cell differentiation in human leukemic (HL-60) cells and insulin signaling (P. Peraldi et al., J. Biol. Chem., 271:13018-13022 (1996)).
In addition to the biological roles of sphingomyelin and ceramide in signal transduction pathways involving cell regulation, recent evidence suggests that sphingomyelinases may be involved in the mobilization of cell surface cholesterol, in cholesterol ester synthesis, and in induction of low density lipoprotein (LDL) receptor activity. See S. Chatterjee, Advances in Lipid Research, 26:25-48 (1993). Recent evidence also supports a possible role of ceramide (a product of N-SMase activity) in programmed cell death and/or "apoptosis" and activation of the gene for nuclear factor (NF)-kB. See A. Alessenko and S. Chatterjee, Mol. Cell. Biochem., 143:169-174 (1995). Sphingomyelinases are also believed to serve as a signal for various exogenous effectors such as antibiotics, drugs, and growth factors, which influence the normal physiology of cells.
A number of specific disorders have been associated with N-SMase. For example, N-SMase has been reported to be associated with insulin resistant diabetes and obesity. See Speigel et al., J. Biol. Chem., 1996. N-SMase is also associated with malaria. The development of the malaria parasite plasmodium requires N-SMase. See Lauer et al., Proc. Nat. Acad. Sci. (USA), 1995. N-SMase also is involved in liver cell proliferation. See Alessenko, Chatterjee, Mol. Cell Biochem., 143:169-174 (1995).
Thus, methods for identifying agents which can modulate N-SMase activity would be highly useful. Moreover, methods for identifying pharmacological agents of interest by automated, high throughput drug screening have become increasing relied upon in a variety of pharmaceutical and biotechnology drug development programs. Unfortunately, however, requisite reagents for such high throughput screening assays to identify agents potentially useful in treatment of N-SMase associated disorders are not readily available. For example, current methods for procuring N-SMase include isolation of the protein from substantial quantities of urine. See, for example, S. Chatterjee, J. Biol. Chem., 264(21):12554 (1989).
It thus would be desirable to have a convenient source of N-SMases. It also would be desirable to have agents that can modulate N-SMase activity. It would be further desirable to have effective assays for identifying compounds that have the potential to modulate N-SMase activity or to diagnose or treat disorders relating to N-SMase.